The production of serine -lactamase is one of the primary resistance mechanisms used by Gram-negative bacterial pathogens against -lactam antibiotics, which include the widely used penicillins and cephalosporins, as well as last resort antibiotics such as the carbapenems. The development of novel -lactamase inhibitors is a pressing need underscored by the continuing mutation of -lactamases. We propose the development of high affinity non-covalent -lactamase inhibitors by targeting conserved structural motifs, particularly those essential for extended spectrum -lactamase activity. Prototypes of these inhibitors have already been identified. Specifically, using the CTX-M Class A -lactamases as a model system, we aim to: 1) apply a fragment-based and structure-guided approach to develop novel -lactamase inhibitor chemotypes; 2) study resistance and ligand binding by ultrahigh-resolution and room-temperature X-ray crystallography; and 3) investigate the evolution of resistance mutations against non-covalent inhibitors. These experiments will lead to new - lactamase inhibitors with clinical potential, while providing a deeper understanding of -lactamase mutations relevant to resistance evolution.